Giardia duodenalis and Its Secreted PPIB Trigger Inflammasome Activation and Pyroptosis in Macrophages through TLR4-Induced ROS Signaling and A20-Mediated NLRP3 Deubiquitination

The extracellular protozoan parasite Giardia duodenalis is a well-known and important causative agent of diarrhea on a global scale. Macrophage pyroptosis has been recognized as an important innate immune effector mechanism against intracellular pathogens. Yet, the effects of noninvasive Giardia infection on macrophage pyroptosis and the associated molecular triggers and regulators remain poorly defined. Here we initially observed that NLRP3 inflammasome-mediated pyroptosis was activated in Giardia-treated macrophages, and inhibition of ROS, NLRP3, or caspase-1 could block GSDMD cleavage, IL-1β, IL-18 and LDH release, and the cell viability reduction. We also confirmed that Giardia-induced NLRP3 inflammasome activation was involved in its K63 deubiquitination. Thus, six candidate deubiquitinases were screened, among which A20 was identified as an effective regulator. We then screened TLRs on macrophage membranes and found that upon stimulation TLR4 was tightly correlated to ROS enhancement, A20-mediated NLRP3 deubiquitination, and pyroptotic signaling. In addition, several Giardia-secreted proteins were predicted as trigger factors via secretome analysis, of which peptidyl-prolyl cis-trans isomerase B (PPIB) independently induced macrophage pyroptosis. This was similar to the findings from the trophozoite treatment, and also led to the TLR4-mediated activation of NLRP3 through K63 deubiquitination by A20. Collectively, the results of this study have significant implications for expanding our understanding of host defense mechanisms after infection with G. duodenalis.

The Roles of Gasdermin D in Coronavirus Infection and Evasion

Pyroptosis is lytic, programmed cell death and plays a critical role against microbial invasion, functioning as an innate immune effector mechanism. The pore-forming protein gasdermin D (GSDMD), a member of gasdermin family proteins, is a primary effector of pyroptosis. The cleavage of inflammasome-associated inflammatory caspases activates GSDMD to liberate the N-terminal effector domain from the C-terminal inhibitory domain and form pores in the cellular plasma membrane. Emerging evidence shows that the pore-forming activity of GSDMD beyond pyroptosis and modifies non-lytic cytosolic protein secretion in living cells and innate immunity. While the essential roles of GSDMD in bacterial infection and cancer have been widely investigated, the importance of GSDMD in virus infection, including coronaviruses, remains elusive. Here, we review the current literature regarding the activation and functions of GSDMD during virus infections. Last, we further discuss the roles of GSDMD and the therapeutic potential of targeting this GSDMD pore-forming activity in coronavirus diseases.

Synovial macrophages in cartilage destruction and regeneration – lessons learnt from osteoarthritis and synovial chondromatosis

Inflammation is a critical process in disease pathogenesis and the restoration of tissue structure and function, for example, in joints such as the knee and temporomandibular. Within the innate immunity process, the body’s first defense response in joints when physical and chemical barriers are breached is the synovial macrophages, the main innate immune effector cells, which are responsible for triggering the initial inflammatory reaction. Macrophage is broadly divided into three phenotypes of resting M0, pro-inflammatory M1-like (referred to below as M1), and anti-inflammatory M2-like (referred to below as M2). The synovial macrophage M1-to-M2 transition can affect the chondrogenic differentiation of mesenchymal stem cells (MSCs) in joints. On the other hand, MSCs can also influence the transition between M1 and M2. Failure of the chondrogenic differentiation of MSCs can result in persistent cartilage destruction leading to osteoarthritis (OA). However, excessive chondrogenic differentiation of MSCs may cause distorted cartilage formation in the synovium, which is evidenced in the case of synovial chondromatosis (SC). This review summarizes the role of macrophage polarization in the process of both cartilage destruction and regeneration, and postulates that the transition of macrophage phenotype in an inflammatory joint environment may play a key role in determining the fate of joint cartilage.

There Is Strength in Numbers: Quantitation of Fc Gamma Receptors on Murine Tissue-Resident Macrophages

Many of the effector functions of antibodies rely on the binding of antibodies/immune complexes to cellular Fcγ receptors (FcγRs). Since the majority of innate immune effector cells express both activating and inhibitory Fc receptors, the outcome of the binding of immune complexes to cells of a given population is influenced by the relative affinities of the respective IgG subclasses to these receptors, as well as by the numbers of activating and inhibitory FcγRs on the cell surface. A group of immune cells that has come into focus more recently is the various subsets of tissue-resident macrophages. The central functions of FcγRs on tissue macrophages include the clearance of opsonized pathogens, the removal of small immune complexes from the circulation and the depletion of antibody-opsonized cells in the therapy of autoimmunity and cancer. Despite these essential functions of FcγRs on tissue-resident macrophages, an in-depth quantification of FcγRs is lacking. Thus, the aim of our current study was to quantify the various Fcγ receptors on macrophages in murine liver, lung, kidney, brain, skin and spleen. Our study identified a pronounced heterogeneity between FcγR expression patterns of the different tissue macrophages, which may reflect their specialized functions within their unique niches in different organ environments.

Ex vivo immune profiling in patient blood enables quantification of innate immune effector functions

The assessment of a patient’s immune function is critical in many clinical situations. In complex clinical immune dysfunction like sepsis, which results from a loss of immune homeostasis due to microbial infection, a plethora of pro- and anti-inflammatory stimuli may occur consecutively or simultaneously. Thus, any immunomodulatory therapy would require in-depth knowledge of an individual patient’s immune status at a given time. Whereas lab-based immune profiling often relies solely on quantification of cell numbers, we used an ex vivo whole-blood infection model in combination with biomathematical modeling to quantify functional parameters of innate immune cells in blood from patients undergoing cardiac surgery. These patients experience a well-characterized inflammatory insult, which results in mitigation of the pathogen-specific response patterns towards Staphylococcus aureus and Candida albicans that are characteristic of healthy people and our patients at baseline. This not only interferes with the elimination of these pathogens from blood, but also selectively augments the escape of C. albicans from phagocytosis. In summary, our model could serve as a valuable functional immune assay for recording and evaluating innate responses to infection.

Calprotectin (S100A8/A9) is an Innate Immune Effector in Experimental Periodontitis

Upregulated in inflammation, calprotectin (complexed S100A8 and S100A9; S100A8/A9) functions as an innate immune effector molecule, promoting inflammation, and as an antimicrobial protein. We hypothesized that antimicrobial S100A8/A9 would mitigate change to the local microbial community and promote resistance to experimental periodontitis in vivo. To test this hypothesis, S100A9 -/- and wild-type (WT; S100A9 +/+ ) C57BL/6 mice were compared using a model of ligature-induced periodontitis. On day 2, WT mice showed fewer infiltrating innate immune cells than S100A9 -/- mice; by day 5, the immune cell numbers were similar. At 5 days post-ligature placement, oral microbial communities sampled with swabs differed significantly in beta diversity between the mouse genotypes. Ligatures recovered from molar teeth of S100A9 -/- and WT mice contained significantly dissimilar microbial genera from each other and the overall oral communities from swabs. Concomitantly, the S100A9 -/- mice had significantly greater alveolar bone loss than WT mice around molar teeth in ligated sites. When the oral microflora was ablated by antibiotic pre-treatment, differences disappeared between WT and S100A9 -/- mice in their immune cell infiltrates and alveolar bone loss. Calprotectin, therefore, suppresses emergence of a dysbiotic, proinflammatory oral microbial community, which reduces innate immune effector activity including early recruitment of innate immune cells, mitigating subsequent alveolar bone loss, and protecting against experimental periodontitis.

Pyroptosis in the Initiation and Progression of Atherosclerosis

Pyroptosis, a newly discovered form of programmed cell death, is characterized by cell swelling, the protrusion of large bubbles from the plasma membrane and cell lysis. This death pathway is mediated by the pore formation of gasdermin D (GSDMD), which is activated by human caspase-1/caspase-4/caspase-5 (or mouse caspase-1/caspase11), and followed with the releasing of both cell contents and proinflammatory cytokines. Pyroptosis was initially found to function as an innate immune effector mechanism to facilitate host defense against pathogenic microorganisms, and subsequent studies revealed that pyroptosis also plays an eventful role in inflammatory immune diseases and tumor resistance. Recent studies have also shown that pyroptosis is involved in the initiation, the progression and complications of atherosclerosis. Here, we provide an overview of the role of pyroptosis in atherosclerosis by focusing on three important participating cells: ECs, macrophages, and SMCs. In addition, we also summarized drugs and stimuli that regulate the progression of atherosclerosis by influencing cell pyroptosis.

Neutrophil Extracellular Traps Serve as Key Effector Molecules in the Protection Against Phialophora verrucosa

Phialophora verrucosa (P. verrucosa) is a pathogen that can cause chromoblastomycosis and phaeohyphomycosis. Recent evidence suggests that neutrophils can produce neutrophil extracellular traps (NETs) that can protect against invasive pathogens. As such, we herein explored the in vitro functional importance of P. verrucosa-induced NET formation. By assessing the co-localization of neutrophil elastase and DNA, we were able to confirm the formation of classical NETs entrapping P. verrucosa specimens. Sytox Green was then used to stain these NETs following neutrophil infection with P. verrucosa in order to quantify the formation of these extracellular structures. NET formation was induced upon neutrophil exposure to both live, UV-inactivated, and dead P. verrucosa fungi. The ability of these NETs to kill fungal hyphae and conidia was demonstrated through MTT and pouring plate assays, respectively. Overall, our results confirmed that P. verrucosa was able to trigger the production of NETs, suggesting that these extracellular structures may represent an important innate immune effector mechanism controlling physiological responses to P. verrucosa infection, thereby aiding in pathogen control during the acute phases of infection.

CD95 expression in triple negative breast cancer blocks induction of an inflammatory state through differential regulation of NF-kB Signaling

CD95L is expressed by tumor-infiltrating lymphocytes to eliminate CD95-expressing tumor cells and thereby CD95 loss by tumor cells is often considered as a consequence of an immunoediting process. Nonetheless CD95 expression is maintained in most triple negative breast cancers (TNBCs), and we recently reported that CD95 loss in TNBC cells triggers the induction of a pro-inflammatory program promoting the recruitment of cytotoxic NK and CD8+ T-cells and impairing tumor growth. Using a comprehensive proteomic approach, we have identified two yet unknown CD95 interaction partners, Kip1 ubiquitination-promoting complex protein 2 (KPC2) and p65. KPC2 contributes to the partial degradation of p105 (NFκB1) and the subsequent generation of p50 homodimers, which transcriptionally represses pro-inflammatory NF-κB-driven gene expression. Mechanistically, KPC2 directly interacts with the C-terminal region of CD95 and links the receptor to RelA (p65) and KPC1, the catalytic subunit of the KPC complex that acts as E3 ubiquitin-protein ligase promoting the partial degradation of p105 into p50. Loss of CD95 in TNBC cells releases KPC2, limiting the formation of the NF-κB inhibitory homodimer complex (p50/p50), promoting NF-κB activation and the production of pro-inflammatory cytokines including CSF1, CSF2, CXCL1 and IL1 members, known to promote recruitment and differentiation of certain adaptive and innate immune effector cells.